High molecular weight extracts of Convolvulus arvensis (field bindweed) and Polygonum convolvulus (wild buckwheat)

ABSTRACT

A bindweed and buckwheat extract is used to inhibit the growth of tumor cells, inhibit the growth of blood vessels, and enhance immune function. The bindweed and buckwheat extract requires minimal purification and produces a low toxicity anti-tumor and immuno-stimulating extract.

This application is a Divisional application of U.S. patent applicationSer. No. 09/597,870, filed Jun. 20, 2000, which is aContinuation-in-part of U.S. patent application Ser. No. 09/249,874,filed Feb. 16, 1999 now U.S. Pat. No. 6,083,510, both of which areherein incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to obtaining purified bindweed and buckwheatextracts by collecting components of a homogenized aqueous solution ofbindweed material. High molecular weight extracts of bindweed werepreviously shown to inhibit the growth of two different types ofvirulent tumors in mice. The extracts possessed immunopotentiatingeffects as evidenced by tumor infiltration by white blood cells in thetumors of treated animals, and induction of lymphocyte proliferation.They further possessed antiangiogenic properties as demonstrated in thechick chorioallantoic membrane assay. The method of extraction of thepresent bindweed and buckwheat extracts is very simple and requires aminimum of purification. The present bindweed and buckwheat extractshave utility as low-toxicity, anti-cancer drugs for human and animaluse.

BACKGROUND OF THE INVENTION

Recently the concept of using biological response modifiers (BRMs) hasbeen considered for the treatment of cancer. Some BRMs are not directlycytotoxic to tumor cells, but possess qualities which change theenvironment of the organism. Immune stimulation and inhibition of newblood vessel growth are two biological response modification strategieswith potential in the treatment of cancer. Examples of immunestimulators with known anti-tumor activity are Polysaccharide K, beta1,3, glucan, and the Maruyama vaccine. All of which containhigh-molecular weight polysaccharides and/or protein. Examples ofangiogenesis inhibiting molecules include TMP-470, and angiostatin,which have demonstrated anti-tumor activity. While investigatingextracts of field bindweed for anti-tumor activity, we initially triedto isolate low-molecular weight alkaloids, which are known to be toxic,and which we suspected of having traditional chemotherapeutic, ortumor-cytotoxic activity.

It was found that the low-molecular weight extracts, containing thetoxic alkaloids, exhibited little anti-tumor activity, while highmolecular weight extracts, which excluded the toxic alkaloids containedsignificant anti-tumor activity by acting as biological responsemodifiers.

SUMMARY OF THE INVENTION

One object of the invention is to provide a pharmaceutical consisting ofhigh molecular weight extracts of field bindweed (Convolvulus arvensis)which have low toxicity to normal cells and induce anti-tumor effects inanimals, inhibit the growth of blood vessels, and enhance immunefunction in mammals. Preferably these high molecular weight extractshave components less than about 500 Daltons removed. More preferably theremoved components are less than about 1,000. Even more preferably, theremoved components are less than about 3,000 Daltons. Even morepreferably, the removed components are less than about 5000, morepreferably 10,000.

One embodiment of the invention is to isolate the high molecular weightextracts of field bindweed (Convolvulus arvensis) using a molecularweight filter or alternatively precipitation with ammonium sulfate.

A further object of the present invention is to provide a method forisolating high molecular weight extracts of field bindweed (Cotvolvulusarvensis), which have low toxicity by virtue of removal of toxiclow-molecular weight components of a crude extract. Preferably thecomponents less than about 3000 Daltons are removed. More preferably thecomponents less than about 6,500 Daltons are removed. Even morepreferably, the components less than about 10,000 Daltons are removed.

One embodiment of the invention is to remove the low molecular weightextracts of field bindweed (Convolvulus arvensis) using a molecularweight filter or alternatively precipitation with ammonium sulfate.

A further object of the present invention is to provide a method for useof high molecular weight extracts of field bindweed (Convolvulusarvensis), which have low toxicity to normal cells and induce anti-tumoreffects in animals.

A further object of the present invention is to provide a method for useof high molecular weight extracts of field bindweed (Convolvulusarvensis), which inhibit new blood vessel growth.

A further object of the present invention is to provide a method for useof high molecular weight extracts of field bindweed (Convolvulusarvensis), which exhibits immunoenhancing effects in animals.

A further object of the invention is a pharmaceutical composition fortreating cancer, inhibiting the growth of new blood vessels, and/orenhancing immune function in a mammal, which is an effective amount ofan aqueous extract of Convolvulus, and a pharmaceutically acceptablevehicle. Preferably the extract is prepared by homogenizing Convolvulusarvensis plant parts and preparing the aqueous extract from the plantparts. Preferably, the plant parts exclude seeds and flowers.Preferably, the extract is prepared using three volumes of water pervolume of homogenized plant parts. Preferably, solids are removed fromthe extract. Preferably, the extract is lyophilized after removal of thesolids.

A further object of the invention is a method for preparing non-toxicextracts of Convolvulus by preparing an aqueous extract of Convolvulus.Preferably, the solid components of the plant are removed.

A further object of the invention is a method for the treatment ofcancer in a mammal by administering a pharmaceutical composition ofConvolvulus in an amount effective to slow or stop the growth of saidcancer.

A further object of the invention is a method for inhibiting bloodvessel growth in a mammal by administering a pharmaceutical compositionof Convolvulus in an amount effective to slow or stop the growth of saidblood vessels.

A further object of the invention is a method for enhancing immunefunction in a mammal by administering the pharmaceutical composition ofConvolvulus in an amount effective to enhance said immune function.Preferably, the immune function is selected from the group consisting oflymphocyte growth and phagocyte activity.

A further object of the invention is a pharmaceutical composition fortreating cancer, inhibiting the growth of new blood vessels, and/orenhancing immune function in a mammal, which is an effective amount ofan aqueous extract of Polygonum, and a pharmaceutically acceptablevehicle. Preferably the extract is prepared by homogenizing Polygonumplant parts; and preparing the aqueous extract from the plant parts.Preferably, the plant parts exclude seeds and flowers. Preferably, theextract is prepared using three volumes of water per volume ofhomogenized plant parts. Preferably, solids are removed from theextract. Preferably, the extract is lyophilized after removal of thesolids.

A further object of the invention is a method for preparing non-toxicextracts of Polygonum by preparing an aqueous extract of Polygonum.Preferably, the solid components of the plant are removed.

A further object of the invention is a method for the treatment ofcancer in a mammal by administering a pharmaceutical composition ofPolygonum in an amount effective to slow or stop the growth of saidcancer.

A further object of the invention is a method for inhibiting bloodvessel growth in a mammal by administering a pharmaceutical compositionof Polygonum in an amount effective to slow or stop the growth of saidblood vessels.

A further object of the invention is a method for enhancing immunefunction in a mammal by administering the pharmaceutical composition ofPolygonum in an amount effective to enhance said immune function.Preferably, the immune function is selected from the group consisting oflymphocyte growth and phagocyte activity.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

We discovered a high molecular weight extract of bindweed thatsurprisingly has low toxicity to normal cells and induces anti-tumoreffects, inhibits the growth of blood vessels, and enhances immunefunction in mammals. We also found that the low molecular weightcomponents of bindweed extract contained alkyloids on the order of300-500 Daltons that were toxic to both normal cells and tumor cells. Inorder to remove the toxic low molecular weight components we used amolecular weight sieve. In general, the method of the invention forobtaining high molecular weight extracts of Convolvulus arvensisconsists of boiling fresh or aged bindweed in an aqueous solution toobtain a brown tea-like mixture. This mixture is then centrifuged orfiltered to remove the solid material to create a solution. It is onlynecessary to remove the 300-500 molecular weight components to removethe toxicity to normal cells. However, we found that the low molecularweight components might nonspecifically stick to the membrane and comeback off. Therefore, preferably, a larger molecular weight sieve isused—on the order of 1000 Daltons, more preferably, 3000 Daltons, evenmore preferably 5000 Daltons. We find that even when molecular weightcomponents of up to 10,000 Daltons are removed, the extract retains itsanti-tumor effects in animals, its ability to inhibit the growth ofblood vessels, and its ability to enhance immune function in mammals. Inthe Examples below, after boiling the fresh or aged bindweed in anaqueous solution to obtain a brown tea-like mixture, the solution isthen passed through a molecular weight filtration device to obtain ahigh molecular weight retentate (BWR), or precipitated using ammoniumsulfate to isolate a high molecular weight precipitate (BWP).Thereafter, the high molecular weight extract is lyophilized, orotherwise concentrated. The extract is subsequently assayed foranti-tumor, immunoenhancing, and anti-angiogenesis activity.

SDS-Page, IEF, and a protein assay, are then used in order to furthercharacterize the components of the extract. The extract is also assayedfor molecular weight using a Superosel2-HR sizing column (PharmaciaBiotech) using known molecular weight standards.

Further features and advantages of the present invention will becomeapparent to those of skill in the art in view of the detaileddescription of the preferred embodiments which follows when consideredtogether with the attached drawings and claims.

Although other material and methods similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are now described.Example 1 describes the steps required to prepare the high molecularweight extracts of bindweed.

EXAMPLE 1 Preparation of High Molecular Weight Extracts of Bindweed

We have isolated high molecular weight extracts of Convolvulus arvensisas follows. Fresh Convolvulus arvensis was harvested and the seeds, andflowers, were discarded. These portions of the plants were homogenizedusing a Waring blender. To the homogenized plant material, 3 volumes ofdeionized water were added to create a mixture. This mixture was thenboiled for 30 minutes to obtain a tea. The solids were removed from thetea by filtration, and the supernatant retained. The supernatant wascooled and from it two high molecular weight isolates were prepared.Some of the tea supernatant was processed in a CH2 concentrator equippedwith a 3,000 or greater Dalton cartridge. The retentate of theconcentrator (containing only molecules larger than 3,000 Daltons) wasretained, and lyophilized, and is herein referred to as BWR.Alternatively, some of the tea supernatant was combined with ammoniumsulfate to yield a final saturation of 50% ammonium sulfate. Thisresulted in precipitation of some of the components of the teasupernatant. The precipitated components were then collected aftercentrifugation at 25,000 g for 30 minutes, and resuspended in phosphatebuffered saline to form a new solution. This new solution was thenpassed through a ultrafiltration device using a 3,000 Dalton membrane.The retentate was collected, and lyophilized, and herein referred to asBWP. BWP, and BWR, were stored at 4° C. in a dessicator prior to use incharacterization, and anti-tumor, anti-angiogenesis, and immuneparameter studies.

In Examples 2 and 3, the high molecular weight extracts werecharacterized and tested for toxicity.

EXAMPLE 2 Characterization of High Molecular Weight Extracts of Bindweed

SDS-Page analysis of BWR and BWP both showed a generalized smearingpattern, which is a polysaccharide characteristic, with occasionalprotein bands at various molecular weights ranging from greater than200,000 Daltons to 6,500 Daltons. Both extracts were positive for theMolish reaction, which indicates the presence of sugar moieties. Andboth were strongly positive for protein using the bicinchroninic acidassay. The above leading to the conclusion that BWP, and BWR containboth protein and saccharide. FPLC chromatography of BWR, and BWP using aSuperose12-HR sizing column demonstrate only high molecular weight peakswith no detection of molecules smaller than 10,000 Daltons.

EXAMPLE 3 Characterization of Toxicity of High Molecular Weight Extractsof Bindweed

Standard chemotherapy screening for cytotoxic anti-tumor agents selectsfor agents with cytotoxic activity at a concentration of 5 mcg/mL. Bothextracts of bindweed were only toxic to cultured tumor cells atconcentrations of 1000 mcg/mL or greater, demonstrating low toxicity.

In Examples 4 through 10 the effects of the high molecular weightextracts, BWR and BWP were analyzed as to anti-angiogenesis activity,anti-tumor activity and effect on immune function.

EXAMPLE 4 Anti-Angiogenesis Activity of BWR

The high molecular weight saccharide/protein containing extracts ofbindweed were prepared in accordance with the above-mentioned methods.

The anti-angiogenesis activity of BWR was examined. Angiogenesis wasinduced on chicken egg chorioallantoic membranes by placing a 2 mmmethylcellulose disc containing 10 mcg of heparin onto 11 day oldchicken egg chorioallantoic membranes. Doses of 200, 100, and 50 mcg ofBWR, were concomitantly added to the heparin-containing disc in threesets of 6 eggs each. 6 eggs served as controls. After 4 days, a largewindow was created over the chorioallantoic membranes, and the numbersof new blood vessels on each egg were counted.

A percent inhibition on angiogenesis was obtained according to thefollowing equation:

Angiogenesis inhibition (%)=(1−Average number of new blood vessels oftest group/Average number of new blood vessels of control group)×100

The results obtained are shown in Table 1 below. It is seen that theangiogenesis, or the induction of new blood vessels was significantlyinhibited by the administration of BWR, in a dose-dependent manner.

TABLE 1 Angiogenesis inhibition 200 mcg/egg 100 mcg/egg 50 mcg/egg 73%55% 18%

EXAMPLE 5 Antitumor Activity of BWR

The antitumor effects of BWR, prepared as described above, wereexamined. Ten 6-week-old mixed-gender Kun Ming mice per group were usedas test animals. S-180 fibrosarcoma cells were subcutaneouslytransplanted to the left inguinal region. Daily for 14 days, BWR wasinjected subcutaneously in the right inguinal region. On the 15th dayafter the transplantation, the tumor was excised and weighed. A percentinhibition on tumor growth was obtained according to the followingequation:

Tumor growth inhibition (%)=(1−Average tumor weight of testgroup/Average tumor weight of control group)×100

The results obtained are shown in Table 2 below. It is seen that tumorgrowth was significantly inhibited by the administration of BWR.

TABLE 2 Average Tumor Growth Dose Tumor Inhibition Group In mg Weight(%) Control 0 1.4 BWR 1 × 14 0.32 77

EXAMPLE 6 Antitumor Effects of BWP

Additionally, the antitumor effects of BWP were examined. Ten 6-week-oldmixed-gender Kun Ming mice per group were used as test animals. S-180cells were subcutaneously transplanted to the left inguinal region.Daily for 14 days, BWP was injected subcutaneously in the right inguinalregion. On the 15th day after the transplantation, the tumor was excisedand weighed. A percent inhibition on tumor growth was obtained accordingto the following equation:

Tumor growth inhibition (%)=(1−Average tumor weight group/Average tumorweight control group)×100

The results obtained are shown in Table 3 below. It is seen that tumorgrowth was significantly inhibited by the administration of BWP.

TABLE 3 Average Tumor Growth Dose Tumor Inhibition Group In mg Weight(%) Control 0 2.5 BWP 1 × 14 0.6 74

EXAMPLE 7 Further Antitumor Effects of BWP

The antitumor effects of BWP were examined further. Ten 6-week-old C57mixed-gender mice per group were used as test animals. LLC, Lewis LungCarcinoma Cells were subcutaneously transplanted to the left inguinalregion. Daily for 21 days, BWR was injected subcutaneously in the rightinguinal region. On the 22^(nd) day after the transplantation, the tumorwas excised and weighed. A percent inhibition on tumor growth wasobtained according to the following equation:

Tumor growth inhibition (%)=(1−Average tumor weight group/Average tumorweight control group)×100

The results obtained are shown in Table 4 below. It is seen that tumorgrowth was significantly inhibited by the administration of BWP.

TABLE 4 Average Tumor Growth Dose Tumor Inhibition Group In mg Weight(%) Control 0 3.5 BWP 1 × 21 1.33 62

EXAMPLE 8 Further Antitumor Effects of BWR

The antitumor effects of BWR were examined further. Ten 6-week-oldmixed-gender Kun Ming mice per group were used as test animals. S-180cells were subcutaneously transplanted to the left inguinal region.Daily for 14 days, BWP was injected intraperitoneally. On the 15^(th)day after the transplantation, the tumor was excised and weighed. Apercent inhibition on tumor growth was obtained according to thefollowing equation:

Tumor growth inhibition (%)=(1−Average tumor weight group/Average tumorweight control group)×100

The results obtained are shown in Table 5 below. It is seen that tumorgrowth was significantly inhibited by the administration of BWR.

TABLE 5 Tumor Average Growth Dose Tumor Inhibition Group In mg Weight(%) Control 0 2.5 BWR 1 × 14 0.8 68

The tumors from this study were embedded in paraffin, stained, andexamined microscopically. It was found that the excised tumors from thetreated group contained large numbers of lymphocytes and monocytes, andonly 10% tumor tissue. The weight of tumor tissue from the tumor tissuein the treated groups was therefore only 10% of that recorded, or 0.08grams rather than 0.8 grams. Therefore the actual inhibition was 96.8%.These results also demonstrate that BWR contains a quality whichenhances the immune response to tumor tissue.

EXAMPLE 9 Effects of BWR and BWP on Human Lymphocyte Growth

The effects of BWR, and BWP on human lymphocyte growth in culture wereexamined. Human lymphocytes were harvested using venipuncture, andsubsequently isolated by use of a centrifuged density gradient. Theywere then incubated in a commercial lymphocyte culture medium (AIM V,containing interleukin 2 and 2 mercaptoethanol) in an atmospherecontaining 95% air, 5% carbon dioxide, at 37° C., for 3 days. Thelymphocytes were then counted using a Coulter Epics XL flow cytometer. Apercent increase in lymphocyte growth was obtained according to thefollowing equation:

Lymphocyte growth increase (%)=(Average number of lymphocytes of testgroup/Average number of lymphocytes of control group×100)−100

The results obtained are shown in Table 6 below. It is seen that BWR,and BWP induced lymphocyte proliferation, in a dose-dependent manner.

TABLE 6 Dose 0 .8  4 20 100  (mcg/ml) BWR 0 0 20  0 35 BWP 0 12 35 20 46

EXAMPLE 10 Effects of BWR and BWP on Human Phagocytic Activity

The effects of BWR, and BWP on human phagocyte activity were examined.Two buffy-coat samples were prepared by centrifuging tubes containinganti-coagulated human blood from two subjects. The samples were thendivided in two. To one buffy-coat from each subject, 2 micrograms ofBWR, and BWP were added. One buffy-coat from each subject served ascontrol. All samples were incubated for 5 hours. Then 30 milligrams offreshly rehydrated baker's yeast was added to all samples. After onehour, a stock 2× solution of acridine orange stain was added to eachsample. An aliquot of each sample was then placed on a microscope slide.The percentage of phagocytes containing intracellular baker's yeast fromeach sample was recorded.

The results revealed an average increase of 85% in the percentage ofphagocytes containing intracellular baker's yeast in the treated samplescompared to the controls. This elucidates another mechanism by which BWRand BWP stimulate the immune system.

The extracts of Example 1 were prepared as shown because it was believedthat the low molecular weight components called alkaloids would beuseful as anti-tumor treatments if a concentration could be found whichwas toxic to cancer cells and minimally toxic to normal cells. When theextract from Example 1 was separated into low and high molecular weightcomponents, it was found that the high molecular weight extract wassurprisingly the part of the extract which inhibited the growth oftumors in mice. Therefore, this extract was used in the experiments setout in Examples 2-10. The low molecular weight molecules were removedbecause it was believed that they would possess molecules calledalkaloids which would be toxic to cells. It is also possible that suchcomponents would be minimally toxic in the context of the completeextract. Therefore, a cruder version of the extract of claim 1 wasprepared in Example 11 and the toxicity tested.

EXAMPLE 11 Crude Water-Soluble Extracts of C. arvensis

Extracts were prepared as in Example 1 with the omission of the step ofremoving the low molecular weight component. The crude water-solubleextracts were prepared as follows:

Fresh Convolvulus arvensis and Polygonum convolvulus were each harvestedand the seeds, and flowers, were discarded. The remaining portions ofthe plants were homogenized using a Waring blender. To the homogenizedplant material, 3 volumes of deionized water were added to create amixture. This mixture was then boiled for 30 minutes to obtain a tea.The solids were removed from the tea by filtration, and the supernatantretained. The supernatant was cooled and lyophilized, and stored at 4°C. in a dessicator prior to use in characterization, and anti-tumor,anti-angiogenesis, and immune parameter studies.

These extracts were tested for toxicity as in Example 3 andsurprisingly, it was found that such extracts were completely nontoxic,exhibiting an LD50 of greater than 20,000 mg/kg. Such a toxicity isequivalent to water. Therefore, the water soluble extract does notresult in a composition with appreciable quantitites of the alkaloidsthat are known to be toxic. This means that the process of preparing theextracts for use in a patient can be simplified considerably, resultingin a much less expensive treatment.

EXAMPLE 12 Extracts of Polygonum convolvulus (Wild Buckwheat)

Although Wild Buckwheat and Field Bindweed are not classified in thesame genus, the two plants have a considerable amount of common traits,including among other things that they are a viney plant, they share asimilar leaf shape, they have common growth traits, etc. It washypothesized that if a Field Bindweed extract possesses biologicalresponse modifiers, or anti-tumor agents, a similar plant would alsopossess such qualities. Therefore, Wild Buckwheat was prepared as inExample 11 and tested in a tumor model identical to that in Example 6.Ten six-week-old mixed gender Kun Ming mice per group were used as testanimals. S-180 cells were subcutaneously transplanted to the leftinguinal region. Daily for 14 days, 50 mg/kg of the Wild Buckwheatextract was injected subcutaneously in the right inguinal region. On the15^(th) day after the transplantation, the tumor was excised andweighted. A percent inhibition was obtained according to the equationgiven in Example 6.

What is claimed is:
 1. A method for inhibiting blood vessel growth in amammal comprising the steps of: administering an aqueous extract of aspecies of Convolvulus in an amount effective to slow or stop the growthof said blood vessels.
 2. The method of claim 1, wherein saidConvolvulus is Convolvulus arvensis.
 3. The method of claim 1, whereinsaid aqueous extract is prepared by a process comprising: homogenizingConvolvulus plant parts; and preparing said aqueous extract from saidplant parts.
 4. The method of claim 3, wherein the plant parts excludeseeds and flowers.
 5. The method of claim 3, wherein the extract isprepared using three volumes of water per volume of homogenized plantparts.
 6. The method of claim 3, wherein solids are removed from saidextract.
 7. The method of claim 6, wherein the extract is lyophilizedafter removal of said solids.
 8. The method of claim 1, wherein saidadministration is by subcutaneous injection.